In nucleic acids (DNA, RNA) which are biological macromolecules, enormous amounts of genetic information essential for vital activities are recorded as sequences composed of combinations of only 4 different bases. Such a nucleic acid allows self-replication using itself as a template by the action of DNA polymerases, and further undergoes processes of RNA polymerase-mediated transcription and ribosome-mediated translation to ensure the transmission of genetic information from DNA to DNA, from DNA to RNA, and/or from RNA to protein. These replication and transmission events of genetic information enabled exclusive base-pairings (A:T/U, G:C). In addition, nucleic acids can form a variety of higher-order structures and hence exert various functions. By way of example, it is one of the indications that a large number of novel nucleic acids having aptamer and/or ribozyme functions have been generated by in vitro selection techniques.
However, unlike proteins which are composed of 20 types of amino acids, the chemical and physical diversity of nucleic acids is limited by the fact that there are only 4 different bases (2 base pairs) in natural nucleic acids. For example, functional RNAs (e.g., tRNA, rRNA, mRNA) found in living organisms utilize various modified bases to stabilize their own structures and/or RNA-RNA and RNA-protein interactions. Thus, it will be very advantageous to expand the repertory of new bases (base pairs) in developing novel functional nucleic acids.
With the aim of further expansion of nucleic acid functions, attempts have been made to design nucleosides or nucleotides having unnatural bases. There are two possible approaches for introducing modified bases (or unnatural bases) into nucleic acids: 1) direct introduction by chemical synthesis; and 2) introduction catalyzed by DNA and RNA polymerase enzymes. In the case of 1), there is a need to solve some problems associated with chemical synthesis, such as the stability of amidite units and the presence of protecting groups appropriate for base moieties. If these problems are solved, various unnatural bases can be introduced in a site-selective manner. However, the nucleic acids thus obtained are difficult to be amplified and it is also difficult to synthesize long-chain nucleic acids. In the case of 2), if the enzymes recognize substrates to cause replication and transcription between artificial base pairs in a complementary manner, nucleic acids containing such artificial base pairs can be amplified and prepared. However, such substrates and base pairs (unnatural nucleotides) are still under development.
Background of Artificial Base Pairs
In natural double-stranded DNA, the “exclusive” A-T and G-C base pairs are formed through specific hydrogen bonding. In recent years, studies have been conducted to develop base pairs that have hydrogen-bonding patterns different from those of natural base pairing and that are capable of eliminating base pairing with natural bases by steric hindrance. For example, Ohtsuki et al. (2001) and Hirao et al. (2002) have designed purine derivatives having a bulky substituent at the 6-position, i.e., 2-amino-6-dimethylaminopurine (x) and 2-amino-6-thienylpurine (s), as well as 2-oxo(1H)pyridine (y) having a hydrogen atom at the site complementary to the bulky substituent, and also have studied x-y and s-y base pairing by the efficiency of Klenow fragment-mediated incorporation into DNA. As a result, the incorporation of y opposite x in the template showed low selectivity, whereas the incorporation of y opposite s showed relatively good selectivity and efficiency (FIG. 1).
The development of the above s-y base pair enabled the selective incorporation of y into RNA. The inventors of the present invention have further conceived that it would be possible to design novel functional molecules such as aptamers and ribozymes once y has been modified to have a functional substituent. Thus, the inventors have developed nucleic acids containing nucleotides, having 5-substituted-2-oxo(1H)pyridin-3-yl bases whose 5-position is substituted with iodine or the like. As to the development history of artificial base pairs, as well as nucleosides and nucleotides having a 5-substituted-2-oxo(1H)pyridin-3-yl group as a base, details can be found in WO2004/007713 (published on Jan. 22, 2004).
Labeling and Detection of Nucleic Acids
Substances such as radioactive elements and fluorescent dyes have previously been used for labeling of nucleic acids. However, nucleic acids containing natural bases have inherent problems; they are labeled only at their ends, or; when any of the labeled natural nucleotides (A, T, G, C) is incorporated, the corresponding bases at a large number of positions are randomly labeled in the inside of their fragments. Thus, it is desired to develop a method for site-specifically labeling the inside of a nucleic acid, e.g., by using a fluorescent dye.
The following documents are listed as reference documents, the entire contents of which are incorporated herein by reference.
Patent Document 1: WO2004/007713
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